A method for activating and amplifying natural killer cells and its use

Abstract

The present invention relates to a method for activating and amplifying natural killer cells and uses thereof. Specifically, the present invention provides a method for activating and culturing NK cells, using a combination of recombinant proteins MICA and MICB as activation reagents to activate and culture NK cells in a coated state. The method of the present invention can achieve efficient amplification of NK cells without the addition of serum and trophoblast cells, and the amplification multiple can be as high as 800 times. The culture process of the present invention is simple, avoids high-risk materials, has high safety, and is particularly suitable for cells from sources such as iPSC-NK. The NK cells finally obtained have high purity and strong killing activity, and can be used to produce high-quality NK cell products.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and in particular to a method for activating and amplifying natural killer cells and a use thereof. Background Art

[0002] In order to achieve better NK expansion, most of the existing NK expansion platforms use expansion methods with serum and trophoblast cells.

[0003] Regarding the method of adding serum, adding human or animal serum to the culture system has the disadvantages of limited serum sources, poor stability between batches, and risks in clinical safety. From a regulatory perspective, although there is no clear ban on the use of serum in cell production processes, its residue and safety assessment require additional attention.

[0004] As for the method using trophoblast cells, trophoblast cells need to be added to the expansion of NK cells. However, trophoblast cells are derived from tumor cells. Although they are inactivated by irradiation or other means before use, the residues of tumor trophoblast cells, including the residues of their degradation products, pose a safety risk in the development of cell therapy products.

[0005] The reported trophoblast-free expansion methods usually use different activation antibodies or ligands to activate NK cells, such as anti-CD2, NKp46 antibodies, etc., and use different cytokines to expand NK cells. However, most of the reported methods use serum, and the performance in terms of NK expansion multiples is not ideal, for example, the NK expansion multiples range from 2.5 times to 200 times.

[0006] Therefore, there is an urgent need in the art to develop a serum-free, trophoblast-free method that can efficiently expand NK cells. Summary of the invention

[0007] The purpose of the present invention is to provide a method for efficiently amplifying NK cells without serum or trophoblast cells.

[0008] In a first aspect of the present invention, a method for expanding natural killer (NK) cells in vitro is provided, comprising the steps of:

[0009] (a) providing a cell culture container, wherein the solid surface of the cell culture container is coated with recombinant proteins MICA and MICB;

[0010] (b) Cultivating NK cells using the cell culture container in step (a).

[0011] In another preferred embodiment, the method does not require the addition of serum or trophoblastic cells.

[0012] In another preferred embodiment, the step (a) comprises: preparing a coating solution, wherein the coating solution comprises recombinant proteins MICA and MICB; and coating the solid surface with the coating solution.

[0013] In another preferred embodiment, the concentration of MICA in the coating solution is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0014] In another preferred embodiment, the concentration of MICB in the coating solution is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0015] In another preferred embodiment, the ratio of MICA to MICB in the coating solution is (1:5) to (5:1).

[0016] In another preferred embodiment, the ratio of MICA to MICB in the coating solution is (1:2) to (2:1).

[0017] In another preferred embodiment, the MICA and MICB are dissolved in a buffer solution to prepare the coating solution.

[0018] In another preferred embodiment, the buffer is selected from the group consisting of phosphate buffer, carbonate buffer, or a combination thereof.

[0019] In another preferred embodiment, the buffer solution is DPBS solution.

[0020] In another preferred embodiment, the volume of the coating solution added to each square centimeter of the solid surface is 0.1-0.5 mL.

[0021] In another preferred embodiment, the volume of the coating solution added to each square centimeter of the solid surface is 0.15-0.35 mL.

[0022] In another preferred embodiment, the volume of the coating solution added to each square centimeter of the solid surface is 0.15-0.25 mL.

[0023] In another preferred embodiment, the protein successfully coated on the solid surface accounts for at least 70% of the total protein content in the coating solution; preferably at least 80%; more preferably at least 90%.

[0024] In another preferred embodiment, the content of MICA successfully coated on the surface of the solid phase carrier is 0.03-3 μg / cm 2 MICB content is 0.03-3μg / cm 2 Preferably, the content of MICA is 0.05-2.5 μg / cm 2MICB content is 0.05-2.5μg / cm 2 More preferably, the content of MICA is 0.1-2 μg / cm 2 MICB content is 0.1-2μg / cm 2 .

[0025] In another preferred embodiment, in step (a), the solid surface is a surface of a cell culture container, and the cell culture container is selected from the group consisting of a culture dish, a multi-well cell plate, a shake flask, or a combination thereof.

[0026] In another preferred embodiment, the solid surface is the surface of magnetic beads in a cell culture container.

[0027] In another preferred embodiment, the coating treatment conditions are: incubation at 4°C±1°C overnight, or incubation at 37°C±3°C for 2-4 hours.

[0028] In another preferred embodiment, the coating treatment conditions are: incubation at 4°C overnight, or incubation at 37°C for 2 hours.

[0029] In another preferred embodiment, the step (b) further comprises:

[0030] (b1) adding culture medium and NK cells to be cultured into the cell culture container in step (a) for activation culture to obtain activated NK cells;

[0031] (b2) transferring the NK cells activated in step (b1) into a culture medium for expansion culture, and harvesting the expanded NK cells.

[0032] In another preferred embodiment, the culture media in step (b1) and step (b2) are the same or different.

[0033] In another preferred embodiment, the culture medium comprises a NK cell culture medium selected from the group consisting of: OptiVitro NK, CTS TM NK-Xpander, Irvine TM PRMIE-XV NK Cell CDM, or a combination thereof.

[0034] In another preferred embodiment, cytokines are also added to the culture medium.

[0035] In another preferred embodiment, the cytokine is selected from the following group: IL-2, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

[0036] In another preferred embodiment, the concentration of the cytokines is: IL-2 (100-2000 IU / ml), IL-12 (5-100 ng / ml), IL-15 (5-100 ng / ml), IL-18 (5-100 ng / ml) and / or IL-21 (10-200 ng / ml).

[0037] In another preferred embodiment, the concentration of the cytokines is: IL-2 (150-1000 IU / ml), IL-12 (10-50 ng / ml), IL-15 (5-50 ng / ml), IL-18 (5-50 ng / ml) and / or IL-21 (10-100 ng / ml).

[0038] In another preferred embodiment, in step (b1), the activated culture is static culture.

[0039] In another preferred embodiment, the activation culture is performed for 2 to 10 days; preferably, 3 to 8 days; more preferably, 4 to 6 days.

[0040] In another preferred embodiment, in step (b1), culture fluid and cytokines are added to the culture medium every 2 to 3 days.

[0041] In another preferred embodiment, in step (b2), the expansion culture is a dynamic culture.

[0042] In another preferred embodiment, in step (b2), the expansion culture is three-dimensional cell culture or dynamic culture on a shaker.

[0043] In another preferred embodiment, in step (b2), culture fluid and cytokines are added to the culture medium every 1 to 3 days.

[0044] In another preferred embodiment, the NK cells are iNK cells differentiated from induced pluripotent stem cells (iPSCs).

[0045] In another preferred embodiment, the NK cells cultured by the method have one or more of the following characteristics:

[0046] (1) Amplification factor ≥ 300 times; preferably ≥ 400 times; more preferably ≥ 700 times;

[0047] (2) Purity ≥ 97%; preferably ≥ 98%; more preferably ≥ 99%;

[0048] (3) When the effector-target ratio E:T = (1:1) to (3:1), the cytotoxicity against target cells is ≥ 50%; preferably ≥ 70%; more preferably ≥ 90%.

[0049] In another preferred embodiment, the target cells are tumor cells.

[0050] In another preferred embodiment, the target cells are K562 and / or Raji cells.

[0051] In the second aspect of the present invention, a use of a recombinant protein combination is provided, wherein the recombinant protein combination is a combination of MICA and MICB, and is used to prepare an activation reagent, wherein the activation reagent is coated into a solid phase carrier and is used for activation and expansion of NK cells.

[0052] In another preferred embodiment, the activation reagent comprises: (i) an effective amount of MICA and MICB; and (ii) a diluent or a carrier.

[0053] In another preferred embodiment, the concentration of MICA in the activation reagent is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0054] In another preferred embodiment, the concentration of MICB in the activation reagent is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0055] In another preferred embodiment, the ratio of MICA to MICB in the activation reagent is (1:5) to (5:1).

[0056] In another preferred embodiment, the ratio of MICA to MICB in the activation reagent is (1:2) to (2:1).

[0057] In another preferred embodiment, the diluent or carrier is selected from the following group: phosphate buffer, carbonate buffer, or a combination thereof.

[0058] In another preferred embodiment, the diluent or carrier is DPBS solution.

[0059] In the third aspect of the present invention, a recombinant protein-coated solid phase carrier is provided, the surface of the solid phase carrier is coated with recombinant protein MICA and recombinant protein MICB, and the solid phase carrier is used to activate and culture NK cells.

[0060] In another preferred embodiment, the solid phase carrier is a cell culture medium or magnetic beads.

[0061] In another preferred embodiment, the cell culture container is selected from the following group: a culture dish, a multi-well cell plate, a shake flask, or a combination thereof.

[0062] In a fourth aspect of the present invention, a kit for activating cultured NK cells is provided, the kit comprising a solid phase carrier coated with the recombinant protein as described in the third aspect of the present invention.

[0063] In another preferred embodiment, the kit further comprises an instruction manual, wherein the instruction manual describes a method for activating and culturing NK cells using a solid phase carrier coated with the recombinant protein.

[0064] In another preferred embodiment, the method comprises the following steps:

[0065] (S1) providing NK cells to be cultured;

[0066] (S2) Activating and culturing the NK cells using the solid phase carrier coated with the recombinant protein.

[0067] In a fifth aspect of the present invention, there is provided a method for preparing a solid phase carrier coated with a recombinant protein as described in the third aspect of the present invention, the method comprising the steps of:

[0068] (Y1) preparing a coating solution, wherein the coating solution comprises recombinant proteins MICA and MICB;

[0069] (Y2) coating the solid surface with the coating solution.

[0070] In another preferred embodiment, the concentration of MICA in the coating solution is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0071] In another preferred embodiment, the concentration of MICB in the coating solution is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0072] In another preferred embodiment, the ratio of MICA to MICB in the coating solution is (1:5) to (5:1).

[0073] In another preferred embodiment, the ratio of MICA to MICB in the coating solution is (1:2) to (2:1).

[0074] In another preferred embodiment, the content of the coating solution added to the surface of the solid phase carrier per square centimeter is 0.1-0.5 mL; preferably 0.15-0.35 mL; more preferably 0.15-0.25 mL.

[0075] In another preferred embodiment, the protein successfully coated on the surface of the solid carrier accounts for at least 70% of the total protein content in the coating solution; preferably at least 80%; more preferably at least 90%.

[0076] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The expansion rate of iNK cells after culture with different activation schemes is shown.

[0078] Figure 2 The fold expansion of iNK cells during culture is shown for the MICA+MICB protein activation regimen compared to the control mAb-CD16 regimen.

[0079] Figure 3 The results show the cytotoxic activity of iNK cells activated and expanded by different schemes against K562 tumor cells, with an effector-target ratio of E:T=1:1.

[0080] Figure 4 The figure shows the direct cytotoxic activity of iNK cells activated and expanded by different schemes against Raji cells (dashed line) and the cytotoxic activity when combined with the antibody Rituximab (solid line).

[0081] Figure 5 The figure shows the expression of CD45, a marker protein on iNK cells, detected by flow cytometry.

[0082] Figure 6 The purity of iNK cells detected by flow cytometry is shown, that is, the proportion of CD3-CD56+ cells. DETAILED DESCRIPTION

[0083] After extensive and in-depth research, the inventors have developed a method for activating and culturing NK cells, using a combination of recombinant proteins MICA and MICB as activation reagents to activate and culture NK cells in a coated state. The method of the present invention can achieve efficient amplification of NK cells without the addition of serum and trophoblast cells, and the amplification multiple can be as high as 800 times. The culture process of the present invention is simple, avoids high-risk materials, has high safety, and is particularly suitable for cells from sources such as iPSC-NK. The NK cells finally obtained have high purity and strong killing activity, and can be used to produce high-quality NK cell products. The present invention was completed on this basis.

[0084] Activation of NK cells

[0085] During the NK cell culture process, it needs to be activated to obtain the ability to expand. The commonly used method for NK cell activation is to add trophoblast cells. Common trophoblast cells include genetically modified chronic myeloid leukemia K562 cells, RPMI8866 and EBV-LCL. These tumor cells have low expression of inhibitory signals and high expression of activation signals on their surfaces. Co-culturing NK cells with them can achieve NK cell activation and large-scale expansion. Compared with the non-trophoblast cell expansion method, the trophoblast cell expansion method can usually expand NK better.

[0086] However, trophoblasts are tumor cells. Although they are inactivated by irradiation or other means before use, the residues of tumor trophoblasts, including the residues of their degradation products, pose safety risks in the development of cell therapy products.

[0087] The activation of NK cells depends on the abnormal expression of MHC molecules and the activation of NK cell surface ligands. There are a variety of activating and inhibitory receptors on the surface of NK cells. These receptors mediate multiple signal pathways by binding to corresponding ligands, respectively realizing the activation and inhibition of NK cells. NK cells need to be fully activated to induce tumor cell killing. Activated NK cells can be amplified in large quantities with high purity and high cytotoxicity.

[0088] The NK cell culture method of the present invention is particularly suitable for iNK cells derived from iPSCs. From the characteristics of NK cells from different sources, iNK cells derived from iPSCs are differentiated from a uniform iPSC working cell bank, and there is no donor difference; in terms of the expression of NK cell surface receptors, there are also differences between iNK cells and NK cells derived from peripheral blood or umbilical cord blood. If iNK cells are activated with activation reagents commonly used in the art that are suitable for NK cells derived from peripheral blood or umbilical cord blood, it is difficult to achieve the desired effect.

[0089] Recombinant MICA and MICB proteins as activation reagents

[0090] MICA / B stands for MHC class I chain-related protein A / B. MICA / B is lowly expressed on normal cells, but upregulated on the surface of damaged, transformed or infected cells. Their binding to NKG2D triggers NK cell effector function. They are two stress-induced ligands of the natural killer (NK) cell receptor NKG2D and play an important role in mediating the cytotoxicity of NK and T cells. In addition, MICA / B is polymorphic, and this polymorphism affects functional responses by regulating its cell surface expression, intracellular trafficking, shedding of immunosuppressive soluble forms, or affinity for NKG2D interaction.

[0091] In the present invention, the use of recombinant MICA and MICB proteins for preparing NK cell activation reagents is provided for the first time. The recombinant MICA and MICB proteins of the present invention are preferably recombinant human MICA proteins and recombinant human MICB proteins. The UniProt ID number of human MICA protein is Q29983, and the UniProt ID number of human MICB protein is Q29980. In a preferred embodiment, the recombinant MICA protein of the present invention comprises an extracellular segment of human MICA, for example, comprising amino acids at positions 24-309 of human MICA protein. In a preferred embodiment, the recombinant MICB protein of the present invention comprises an extracellular segment of human MICB, for example, comprising amino acids at positions 23-308 of human MICB protein.

[0092] In the present invention, MICA and MICB proteins are coated on the surface of a cell culture container to culture NK cells, which can significantly activate the proliferation ability of NK cells and improve the expansion rate of NK cells during the culture process.

[0093] In the present invention, the combination of MICA and MICB has a synergistic effect on the activation of NK cells, that is, the effect of MICA protein or MICB protein alone on the activation of NK cells is significantly higher.

[0094] In addition, the present invention also found that MICA and MICB proteins have a significant effect on the proliferation of NK cells only in the coated state, but have no significant activation effect in the solution state. Therefore, MICA and MICB proteins can be coated on the surface of cell culture containers, such as culture dishes and multi-well plates, for subsequent NK cell culture; MICA and MICB proteins can also be coated on the surface of magnetic beads for NK cell culture.

[0095] NK cell activation and culture method of the present invention

[0096] In the present invention, a method for activating and culturing NK cells using recombinant proteins MICA and MICB as activation agents is provided.

[0097] As used herein, the term "activation" means that during the NK cell culture stage, NK cells have a higher expansion rate, and the harvested NK cells have high purity and high cell killing activity.

[0098] The recombinant proteins MICA and MICB of the present invention can only exert activation effects in a coated state. First, a coating solution containing both MICA and MICB proteins is prepared, wherein the concentration of MICA in the coating solution is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml; the concentration of MICB is 0.1-15 μg / ml; preferably 0.5-10 μg / ml; more preferably 1-5 μg / ml.

[0099] Preferably, the ratio of MICA to MICB in the coating solution is (1:5) to (5:1); more preferably (1:2) to (2:1). In a preferred embodiment, a ratio of MICA to MICB of 1:2, 1:1 or 2:1 has a higher activation level.

[0100] After coating the surface of the cell culture container with the coating solution, NK professional culture medium is added to culture NK cells. In the present invention, the type of NK culture medium is not limited, preferably a serum-free NK culture medium. Preferably, a cytokine combination is added to the culture medium, such as IL-2 (100-2000 IU / ml), IL-12 (5-100 ng / ml), IL-15 (5-100 ng / ml), IL-18 (5-100 ng / ml) and IL-21 (10-200 ng / ml).

[0101] Preferably, the NK cell activation culture process of the present invention mainly includes two stages: an activation culture stage with the addition of MICA and MICB, and an expansion culture stage without the addition of an activator.

[0102] In the activation culture stage, NK cells are inoculated into a container coated with MICA and MICB and statically activated in a culture medium. In a preferred embodiment, this stage lasts for 5 days, and an equal volume of culture medium and cytokines are added on the 3rd day. Then, the expansion culture stage is entered.

[0103] In the expansion culture stage, no activation reagent is added, and NK cells are expanded and cultured using three-dimensional cell culture and / or on a shaker. The culture medium used in this stage is the same as the activation stage. This process lasts for 10 to 12 days, and the culture medium and cytokines are supplemented every 1 to 3 days depending on the cell growth. NK cells are harvested.

[0104] Optionally, the harvested NK cells are tested for expansion multiple, purity, cell killing activity, etc.

[0105] The activation culture method of the present invention can harvest NK cells with high expansion multiples, high purity and high cell killing activity, and no serum and trophoblast cells are added in the process, so the process is simple and safe.

[0106] The main advantages of the present invention include:

[0107] 1. The NK cell activation and expansion method of the present invention can obtain NK cells with a high expansion multiple, and the highest expansion multiple can reach 800 times.

[0108] 2. The NK cells obtained by the NK cell activation and expansion method of the present invention have high purity and high cell killing activity.

[0109] 3. The NK cell activation and expansion method of the present invention is serum-free and trophoblast-free, thus avoiding the risk of introduction of animal-derived factors brought by materials and increasing the safety of the product.

[0110] 4. The NK cell activation and expansion method of the present invention has a simple process flow and reduces the cost of quality supervision;

[0111] 5. The NK cell activation and expansion method of the present invention is generally applicable to a variety of NK cells, especially to iPSC-derived iNK cells, and the effect is consistent and stable in different batches of iNK cells.

[0112] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0113] Materials and Methods

[0114] 1. Materials

[0115] 1. Culture medium

[0116] In the embodiments of the present invention, a serum-free NK-specific culture medium, OptiVitro NK culture medium, is used. The main components of the culture medium include amino acids, vitamins, trace elements, inorganic salts, and human albumin.

[0117] 2. NK cells

[0118] In the embodiment of the present invention, iNK cells obtained by cell differentiation process from reprogrammed iPSC cells are used as the source of NK cells for expansion and culture.

[0119] 3. Activation Reagents

[0120] In the embodiments of the present invention, recombinant MICA and / or recombinant MICB proteins are used as NK cell activators, and CD16 monoclonal antibodies are used as control activators. Recombinant MICA protein (Acro Biosystems, catalog number: MIA-H5254), recombinant MICB protein (Acro Biosystems, catalog number: MIB-H52H3), and mAb-CD16 (Excellbio, catalog number: BA0112) are all commercially available recombinant proteins or antibodies.

[0121] NK cell activation culture program

[0122] Scheme 1. Activation and expansion of NK cells using MICA+MICB coating solution

[0123] (1) Prepare coating solution. Use DPBS as diluent to dissolve recombinant MICA protein and recombinant MICB protein to a concentration of 1-3 μg / ml, respectively, to obtain MICA+MICB coating solution with different concentration ratios. The concentration combinations are as follows: MICA (2 μg / ml) + MICB (1 μg / ml), MICA (1 μg / ml) + MICB (2 μg / ml), MICA (1.5 μg / ml) + MICB (1.5 μg / ml). Add the mixed coating solution to the culture dish with untreated (NTC) surface, cover the surface with the liquid, and incubate at 4°C overnight or 37°C for 2 hours.

[0124] (2) Resuspend the iNK cells differentiated from iPSCs to a density of 2-10E5 using serum-free NK-specific culture medium, and add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10 ng / ml), IL-18 (concentration 5 ng / ml), and IL-21 (concentration 10 ng / ml) to the culture medium.

[0125] (3) Take out the coated culture plate, remove the coating solution, add the iNK cell suspension with stimulating factors to the coated culture plate, incubate in a 37°C, 5% CO2 incubator for 3 days, and activate by static activation.

[0126] (4) After 3 days, add half the volume of fresh culture medium relative to the previous culture system, and continue to add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10 ng / ml), IL-18 (concentration 5 ng / ml), and IL-21 (concentration 10 ng / ml), and then let it rest for activation for another 2 days.

[0127] (5) After a total of 5 days of activation culture, the activated iNK cells were transferred to a shake flask for expansion culture. While transferring, fresh culture medium was added to dilute the activated cells to a density of 2~10E5, and IL-2 (concentration 200IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10ng / ml), IL-18 (concentration 5ng / ml), and IL-21 (concentration 10ng / ml) were added. The shake flask was placed in a shaker and cultured dynamically at 45rpm.

[0128] (6) The culture medium and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10 ng / ml), IL-18 (concentration 5 ng / ml), and IL-21 (concentration 10 ng / ml) were then added every 1 to 3 days, and the culture was continued for 10 to 15 days. After washing and freezing, the iNK product was obtained.

[0129] Scheme 2. Activation and expansion of NK cells using MICA or MICB coating solution alone

[0130] The coating solution in step (1) was prepared as a coating solution containing only MICA or only MICB using the method of Scheme 1. The concentrations of the coating solutions were: MICA (1 μg / ml), MICA (2 μg / ml), MICA (3 μg / ml), MICB (1 μg / ml), MICB (2 μg / ml), and MICB (3 μg / ml). The other steps were the same as Scheme 1.

[0131] Scheme 3. Activation and expansion of NK cells with soluble MICA+MICB solution

[0132] The method of Scheme 1 was adopted, except that the coating in step (1) was replaced by directly adding soluble MICA (1.5 μg / ml) + MICB (1.5 μg / ml) solution to the culture medium. The other steps were the same as those in Scheme 1.

[0133] Protocol 4. mAb-CD16 activation and expansion of NK cells

[0134] The method of Scheme 1 was adopted, except that the coating in step (1) was replaced by directly adding mAb-CD16 (3 μg / ml) solution into the culture medium. The other steps were the same as those in Scheme 1.

[0135] 3. Detection and analysis of NK cell proliferation

[0136] 1. NK cell expansion efficiency detection

[0137] During the expansion culture phase after activation culture, the cell growth status was recorded, and at different time points of culture (such as the 5th, 7th, 9th, 11th, 13th, and 15th days of expansion), the cells were counted and the NK cell expansion curve was drawn.

[0138] 2. NK cell cytotoxicity assay

[0139] After the amplified NK cells were co-cultured with the target cells K562 cells, flow cytometry was used to detect the killing effect of NK on target cells (Annexin V-7AAD- represents surviving target cells) at different E:T effector-target ratios (NK cells: target cells).

[0140] 3. NK cell flow cytometry analysis

[0141] Take the NK cells that are being amplified or activated and amplified, and use flow cytometry to detect the NK cell surface antigen phenotype (CD3, CD16, CD56) and the purity of the cells (CD3-CD56+%).

[0142] Example 1: Comparison of the effects of different culture schemes on activating and expanding NK cells

[0143] Under the same conditions, iNK cells were activated and expanded using methods 1 to 4, and a negative control group was set up to culture without adding activators (No-activation). The expansion multiples were detected on the 11th day after culture, and the data of other groups were normalized with the results of the mAb-CD16 group. The results are shown in Figure 2. Figure 1 shown.

[0144] The results showed that without adding activators (No-activation), NK had basically no expansion ability; the combination of recombinant MICA and recombinant MICB proteins had the strongest activation ability at different concentration combinations, and the activation effect was equivalent when the ratio of MICA to MICB was 1:2, 1:1 or 2:1, which was significantly better than the positive control mAb-CD16 group.

[0145] In addition, the activation effect of recombinant MICA or recombinant MICB alone was poor, especially the group using MICB alone, which was lower than the mAb-CD16 group; but the combination of MICA+MICB was the most significant, indicating that the combination of MICA+MICB has an unexpected synergistic effect on the activation and expansion of iNK cells.

[0146] Figure 1 The results also showed that recombinant MICA and recombinant MICB proteins had the effect of activating NK cells only when they were coated on the culture plate, and could not activate NK cells when only added to the culture medium.

[0147] Example 2: Activation effect of the solution of the present invention

[0148] Existing studies have shown that mAb-CD16 has a good effect on activating and expanding NK cells. In the present invention, mAb-CD16 is used as a positive control to illustrate the activation effect of the recombinant MICA+MICB combination of the present invention on NK.

[0149] Comparison of the effects of mAb-CD16 group and MICA (1.5 μg / ml) + MICB (1.5 μg / ml) group on iNK cell expansion Figure 2 As shown. The results showed that the iNK cells cultured with the recombinant MICA+MICB protein combination had significantly better expansion ability than CD16 monoclonal antibody after the 8th day of culture. On the 11th day, the expansion multiple of the NK cells of the recombinant MICA+MICB protein combination reached about 800 times, while that of the mAb-CD16 group was less than 600 times. This shows that the activation effect of MICA+MICB protein is better than that of the commonly used activation reagent mAb-CD16 in the field.

[0150] Example 3: Cytotoxic activity of NK cells after expansion culture

[0151] Tumor cells K562 or Raji were used as target cells to detect the cell killing activity of NK cells cultured with mAb-CD16 and NK cells cultured with the combination of MICA+MICB of the present invention.

[0152] The results are as follows Figure 3 and Figure 4 The results showed that NK activated by recombinant MICA and recombinant MICB proteins had high cytotoxicity against tumor cells such as K562, and combined with antibodies, it could further enhance the cytotoxicity against Raji cells through the ADCC effect.

[0153] The above results indicate that the addition of MICA and MICB during the culture stage enables NK cells to retain a high level of cell-killing activity.

[0154] Example 4: Flow cytometry detection of NK cell phenotype and purity after amplification and culture

[0155] NK cells that are being amplified or activated are taken and the surface antigen phenotype of NK cells is detected by flow cytometry. CD45 is a marker protein for the phenotype of iNK cells after amplification. The purity of the cells is also detected and characterized by the percentage of CD3-CD56+ cells.

[0156] The results are as follows Figure 5 and Figure 6 shown. Figure 5 It showed that the NK cells obtained by both mAb-CD16 and MICA+MICB activation methods were 100% CD45+ iNK cells. Figure 6 The proportion of CD-CD56+ cells in the sample indicated the purity of iNK cells. The results showed that the purity of iNK obtained by the MICA+MICB activation scheme of the present invention was 99.2%, which was greater than that of the control mAb-CD16.

[0157] The above results indicate that the method of activating and culturing iNK cells using a combination of MICA and MICB in the present invention can obtain high-purity iNK cells for use in preparing high-quality iNK products.

[0158] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for in vitro expansion of natural killer (NK) cells, characterized in that: Includes steps: (a) providing a cell culture container, wherein the solid surface of the cell culture container is treated with a coating solution, and the coated recombinant proteins are MICA and MICB; the concentration of MICA in the coating solution is 0.1-15 μg / ml, and the concentration of MICB is 0.1-15 μg / ml; and the ratio of MICA to MICB in the coating solution is (1:5) to (5:1); (b) culturing NK cells using the cell culture container in step (a); Furthermore, the method does not require the addition of serum or trophoblast cells.

2. The method according to claim 1, characterized in that Step (a) comprises: preparing a coating solution; coating the solid surface with the coating solution, wherein the volume of the coating solution added to each square centimeter of the solid surface is 0.1-0.5 mL.

3. The method according to claim 1, characterized in that The concentration of MICA in the coating solution is 0.5-10 μg / ml; the concentration of MICB in the coating solution is 0.5-10 μg / ml.

4. The method according to claim 1, characterized in that The ratio of MICA to MICB in the coating solution is (1:2) to (2:1).

5. The method according to claim 1, characterized in that Step (b) further comprises: (b1) adding culture medium and NK cells to be cultured into the cell culture container in step (a) for activation culture to obtain activated NK cells; (b2) transferring the NK cells activated in step (b1) into a culture medium for expansion culture, and harvesting the expanded NK cells.

6. The method according to claim 5, characterized in that Cytokines are also added to the culture medium, and the cytokines are selected from the following group: IL-2, IL-12, IL-15, IL-18, IL-21 or a combination thereof.

7. The method according to claim 1, characterized in that The NK cells are iNK cells differentiated from induced pluripotent stem cells.

8. The method according to claim 1, characterized in that The NK cells cultured by the method have one or more characteristics selected from the following group: (1) Amplification factor ≥ 300 times; (2) Purity ≥ 97%; (3) When the effector-target ratio E:T = (1:1) ~ (3:1), the killing activity against target cells is ≥ 50%.

9. Use of a recombinant protein-coated solid phase carrier, characterized in that: A kit for preparing activation and cultured NK cells; the recombinant proteins coated on the surface of the solid phase carrier are MICA and MICB, and the solid phase carrier is used to activate and culture NK cells; the solid phase carrier is prepared by the following method: providing a coating solution, the concentration of MICA in the coating solution is 0.1-15μg / ml, the concentration of MICB is 0.1-15μg / ml, and the ratio of MICA to MICB in the coating solution is (1:5) to (5:1); the surface of the solid phase carrier is coated with the coating solution, and the volume of the coating solution added to the surface of the solid phase carrier per square centimeter is 0.1~0.5 mL.

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